Mass spectrometry for quality control of bispecific antibodies after SDS-PAGE in-gel digestion.

Abstract

Recombinant bispecific antibodies (bsAbs) are increasingly included in regimens for cancer therapy. Strict good manufacturing practice (GMP) compliant quality control measures are required to ensure quality and safety of these innovative biologicals. Gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and size exclusion chromatography (SEC) are the cornerstones of quality control methods. BsAbs are often prone to aggregation or incomplete synthesis due to their artificial nature. In addition, host cell proteins and host cell DNA as well as impurities from the purification process itself constitute potential contaminants. Such impurities may then appear as additional, unexpected bands or peaks on SDS-PAGE gels and SEC, respectively. Here we describe a standardized protocol for rapid analysis of recombinant antibodies by mass spectrometry (MS) after tryptic digestion of bands excised from SDS-PAGE gels. We have used this protocol to characterize unexpected "contaminating bands" that were observed during the clinical development of a novel bsAb with PSMAxCD3 specificity, either during the production of the protein itself or during the development of a surrogate molecule for evaluation in syngeneic mouse models. MS analysis allowed us to precisely determine the origin of these bands, which resulted from artifacts or from incomplete protein synthesis. The combined utilization of SDS-PAGE und MS can therefore substantially support GMP-compliant production of recombinant proteins.